CD38 catalyzes the hydrolysis of nicotinamide adenine dinucleotide (NAD+) producing ADP-ribose (ADPR) and also catalyzes cyclization of NAD+ to produce cyclic ADP-ribose (cADPR). Both ADPR and cADPR are physiological messengers required for calcium mobilization and motility of antigen presenting cells involved in the etiology of autoimmunity and inflammation [Perraud et al. (2001) Nature 411: 595-599; Sano et al. (2001) Science 293: 1327-1330; Clapper et al. (1987) J. Biol. Chem. 262: 9561-9568; Partida-Sanchez et al. (2004) J. Immunol. 172: 1896-1906; Partida-Sanchez et al. (2004) Immunity 20:279-291]. CD38/cADPR calcium mobilization has also been shown to have a role in airway hyper-responsiveness (Deshpande et al., (2003) FASEB Journal 17: 452-454. Therefore, potent, specific inhibitors of CD38 appear to be useful in the treatment of autoimmune and inflammatory diseases and diseases associated with hyper-reactivity of the airways, such as asthma. A robust, accurate, high-throughput assay for the identification of CD38 inhibitors would therefore be highly desirable.
Such an assay has not been described previously. Assays for CD38 catalytic activity were either tedious (low throughput) or utilized non-physiological substrates, and involved inefficient detection means that were not amenable to accurate identification of hits from large, diverse compound libraries (≧10,000 compounds). For example, work published by Wall [Wall et al. (1998) Biochem. J. 335: 631-636] and Higashida [Higashida et al. (2000) Biochem. J. 352: 197-202] utilized radioactively labeled NAD+ substrate, with analysis and measurement of reaction products by scintillation counting, high pressure liquid chromatography (HPLC) and autoradiography. These methods are low-throughput, labor intensive, and environmentally unfriendly.
Muller-Steffner and colleagues published work in 1992[Muller-Steffner et al. (1992) J. Biol. Chem. 267: 9606-9611], describing data derived from a protocol which utilized a non-physiological NAD+ substrate analog 1,N6-etheno NAD+. CD38 glycohydrolase activity yields the fluorescent product 1,N6-etheno ADP-ribose (excitation λ=310 nm; emission λ=410 nm), which has enhanced fluorescence compared to the unhydrolyzed, non-physiological substrate. Graeff and colleagues [Graeff et al. (1994) J. Biol. Chem. 269: 30260-30267] published data derived from a similar protocol in which a second, non-physiological substrate, NAD+ analog, nicotinamide guanine dinucleotide (NGD+), was utilized to assay for CD38 cyclase activity. The product formed in this reaction, cyclic GDP-ribose (cGDPR), was fluorescent and could be monitored with a spectrometer (excitation λ=300 nm; emission λ=410 nm). In 2003 Graeff and Lee [Graeff & Lee (2003) Comb. Chem. & High Through. Screen. 6: 367-379] provided details for a third fluorescence based assay constructed to take advantage of CD38 cADPR hydrolytic activity. This assay monitors CD38 dependent hydrolysis of the fluorescent cADPR analog, cyclic inosine diphosphate ribose (cIDPR). As cIDPR is hydrolyzed by CD38, loss of fluorescent signal is monitored with a spectrometer (excitation λ=310 nm; emission λ=410 nm). In the aforementioned assays, the detection wavelength was below 450 nm, a range in which many compounds in today's modern, diverse libraries absorb light, thus leading to spectral interference and erroneous results (false positive data).